JP6139259B2 - Method for producing wound healing agent - Google Patents

Description

The present invention relates to a method for producing a wound therapeutic agent with enhanced wound healing effect.

In recent years, the existence of various factors that promote healing in the exudate of wounds such as cuts, scratches, burns, etc. has been clarified, and as a method for treating wounds, the wound is kept wet with exudate, Research on so-called wet dressing is underway.
As such a wet dressing material, for example, an amorphous gel wound dressing of cellulose derived from microorganisms has been proposed in addition to an adhesive polyurethane film, hydrocolloid and the like (for example, Patent Document 1).

The microorganism-derived cellulose amorphous gel wound dressing of Patent Literature 1 is biosynthetic cellulose produced by cellulose-producing organisms such as Acetobacter xylinum, which is non-pyrogenic by a series of chemical washing with sodium hydroxide and hydrogen peroxide. Given a cellulose film, this film is then wet milled to prepare an amorphous gel shape with a cellulose content of about 4% or about 7% by weight.
The microorganism-derived cellulose amorphous gel wound dressing of Patent Document 1 not only enhances the water supply ability as a water supply source for supplying water to the wound, but also has a body fluid absorption capacity for absorbing body fluid from the wound bed that has exuded. Can be achieved at the same time.

JP 2006-512981 A (paragraphs 0015 to 0016, etc.)

However, there has been a demand for development of a wound healing agent such as the microorganism-derived cellulose amorphous gel wound dressing of Patent Document 1 that has a higher effect of promoting wound healing.
This invention is made | formed in view of said subject, The objective of this invention is providing the manufacturing method of the novel wound healing agent with a high healing promotion effect of a wound.
Another object of the present invention is to provide a method for producing a wound therapeutic agent having a high healing promotion effect in the initial stage of the wound healing process.

As a result of diligent research, the present inventors have surprisingly found that Euglena-derived paramylon has a high wound healing promoting effect as compared with conventionally known wound healing promoting agents, and reached the present invention. .
In addition, the wound healing process has overlapping periods of inflammation, proliferation, and maturation, and neutrophils and monocytes / macrophages that wet the damaged part play an important role in the inflammation (Yasunori Sato, “Expression of Interleukin (IL) -10 in Skin Wound Healing Process and Its Forensic Application”, Kanazawa University Full Medical Journal, Vol. 108, No. 4, p.486-502).
And Euglena-derived paramylon has been found to act particularly on neoplastic and inflammatory macrophages and promote inflammatory cytokine secretion.

Therefore, according to the present invention , according to the present invention , a paramylon solution obtained by dissolving paramylon derived from Euglena in a paramylon-soluble solvent at a temperature of 60 to 70 ° C. is cooled to 40 to 50 ° C. This is solved by a method for producing a wound therapeutic agent, which is produced by passing a paramylon-soluble solvent through a solvent that is a solvent and capable of extracting the paramylon-soluble solvent .
By comprising in this way, the wound therapeutic agent which can be applied directly to an affected part and is easy to use can be obtained.
According to the present invention, a wound treatment agent containing Euglena-derived paramylon as an active ingredient can be obtained.
Wound wound healing agent may be a therapeutic agent for wounds in the healing process including the time to inflammatory phase immediately after injury.
Since the effect of promoting the secretion of cytokine by paramylon is remarkable in the period from immediately after injury to the inflammatory phase, particularly in the early stage of the inflammatory phase, this configuration can efficiently promote wound healing. .

Moreover, it is good also as a wound therapeutic agent which accelerates | stimulates the dermis surrounding the wound in the wound healing process and promotes the formation of the dermis around the wound.
It is empirically known that wound healing is rapid when the dermis around the wound is prominent. By configuring as such a wound therapeutic agent, healing is promoted from the initial stage of the healing process, and early healing of the wound becomes possible. Therefore, it becomes easy to realize that the healing is progressing, and the physical and mental burden is reduced. At the same time, chain wound site symptoms are prevented from worsening, the influence on other organs and tissues of the body is reduced, and a general healing effect is obtained.

It may be a wound therapeutic agent that promotes cytokine secretion in the wound healing process.
By configuring in this way, it is possible to promote wound healing in the inflammatory phase where neutrophils and macrophages are largely involved in the wound healing process, and healing is promoted from the early stage of the healing process, enabling early wound healing. . Therefore, it becomes easy to realize that the healing is progressing, and the physical and mental burden is reduced. At the same time, chain wound site symptoms are prevented from worsening, the influence on other organs and tissues of the body is reduced, and a general healing effect is obtained.

Wound wound healing agent may be a diabetic wound care agent.
In diabetic patients, cell migration becomes dull, secretion of cytokines is suppressed, because it may or may not proceed or delay the progress of wound healing, a wound treatment agent to be manufactured in this invention diabetics When applied to a wound, cytokine secretion can be promoted and wound healing can proceed.

Also, a wound therapeutic agent, may is a skin external preparation.

According to the method for producing a wound treatment agent containing Euglena-derived paramylon of the present invention as an active ingredient, a wound treatment agent having a high wound healing promoting effect can be obtained even when compared with conventionally known wound healing promoting agents.
In addition, wound healing can be promoted in the inflammatory phase where neutrophils and macrophages are largely involved in the wound healing process, healing is promoted from the early stage of the healing process, and early wound healing is possible. Therefore, it becomes easy to realize that the healing is progressing, and the physical and mental burden is reduced. At the same time, chain wound site symptoms are prevented from worsening, the influence on other organs and tissues of the body is reduced, and a general healing effect is obtained.

3 is an experimental protocol of Test Example 1. In Experiment 1, it is explanatory drawing which shows arrangement | positioning of the defect wound produced in the mouse | mouth, the applied film, etc. FIG. 6 is a graph showing changes in body weight of mice in each group in Test Example 1. 6 is a graph showing changes in the wound area ratio of mice in each group in Test Example 1. It is a graph which shows the change of the dermal area around a wound of the mouse | mouth of each group of the wound creation 0th day in Experimental example 1. FIG. 2 is a photograph showing the appearance of the wound of each group of mice on Day 0 of wound preparation in Test Example 1. FIG. It is a photograph which shows the external appearance of the wound of the mouse | mouth of each group 1 day after wound preparation in Test Example 1. FIG. It is a photograph which shows the external appearance of the wound of the mouse | mouth of each group 3 days after wound preparation in Test Example 1. FIG. It is a photograph which shows the external appearance of the wound of the mouse | mouth of each group 5 days after wound preparation in Test Example 1. FIG. It is a graph which shows the amount of IL-6 in blood of the mouse | mouth of a control group of wound preparation in Test Example 1 on the 0th, 3rd, and 5th days. It is a graph which shows the amount of blood IL-6 of the mouse | mouth of each group 3 days after wound preparation in Experiment 1, and 5 days after. 3 is a graph showing the amount of IFN-γ in blood of mice in a control group on day 0, 3, and 5 days after wound creation in Test Example 1. FIG. It is a graph which shows the amount of IFN-γ in blood of each group of mice 3 days and 5 days after wound creation in Test Example 1. 2 is a graph showing blood VEGF levels in mice of a control group on day 0, 3, and 5 days after wound creation in Test Example 1. FIG. 3 is a graph showing blood VEGF levels in mice of each group 3 days and 5 days after wound creation in Test Example 1.

Hereinafter, the manufacturing method of the wound therapeutic agent which concerns on embodiment of this invention is demonstrated.
Method for producing a wound treatment agent of the present invention relates to a method for producing a wound therapeutic agent as a main component the paramylon from Euglena.
In this specification, a wound refers to a wound or ulcer that is a physical damage to a surface tissue such as a skin tissue caused by an external or internal factor, and is a wound, cut, abrasion, burn, contusion, laceration, or bite wound. , Pressure ulcers, diabetic ulcers, skin ulcers and the like.
The “Euglena” of the present invention is a component that includes all plants classified as Euglena in zoological or botanical classification, varieties thereof, and variants thereof, and has an inhibitory action on α-glucosidase activity. Means all plants including
Here, Euglena microorganisms in the zoology are euglenoidina subgenus of Euglenida belonging to Protozoa's Mastigophora and Phytastigophorae. It is a microorganism belonging to the eye (Euglenoidina). On the other hand, microorganisms belonging to the genus Euglena belong to the order of Euglenaes belonging to Euglenophyceae in Euglenophyta in botany.

  Microorganisms of euglenoids, specifically, Euglena acus, Euglena caudata, Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena intermedia, Euglena mutabilis, Euglena oxyuris, Euglena proxima, Euglena spirogyra, Euglena viridis, Euglena vermiformis Etc. Of these, Euglena gracilis, which is widely used for research, is particularly suitable.

Euglena can be cultured using a Cramer-Myers medium, a Hutner medium, a Koren-Hutner medium, a modified medium in which a partial composition thereof is changed, or the like. A Sakaguchi flask, an Erlenmeyer flask, a reagent bottle, etc. can be used for a culture container. Since Euglena to assimilate CO _2, when cultured using the Cramer-Myers medium is autotrophic medium is preferably be passed through into the medium air containing 1 to 5% CO _2. Further, in order to sufficiently develop the chloroplast, about 1 to 5 g of ammonium phosphate may be added per liter of the medium. The culture temperature is usually 20 to 34 ° C., particularly preferably 28 to 30 ° C. Depending on the culture conditions, Euglena usually enters a logarithmic growth phase 2 to 3 days after the start of the culture, and reaches a stationary phase about 4 to 5 days.
Euglena may be cultured under light irradiation (light culture) or non-irradiated (dark culture).

Paramylon is a polymer obtained by polymerizing about 700 glucose by β-1,3-linkage, and is a storage polysaccharide contained in Euglena. Paramylon can be accumulated in cells by culturing Euglena on a medium mainly composed of glucose. Paramylon in Euglena cells takes a particulate form with a diameter of several μm in the cell, and can be easily taken out by crushing the cells, and can be purified by treatment with alcohol or toluene.
In the present invention, paramylon powder is used for the preparation of a wound therapeutic agent, but it may be treated with alkali treatment, chemical modification, cross-linking or the like, or a water-soluble paramylon derivative.

The wound therapeutic agent produced in the present invention may be an external preparation for wound treatment, and the external preparation for wound treatment may be a paramylon film in the form of a film.
The paramylon film as one embodiment of the wound treatment agent produced in the present invention is prepared by dissolving paramylon in a paramylon-soluble solvent at a temperature of room temperature to 80 ° C., preferably 60 to 70 ° C. And this paramylon solution is made to pass through a solvent that is a poor solvent for paramylon and from which a paramylon-soluble solvent can be extracted.
In the method for producing a paramylon film, first, paramylon is dissolved in a paramylon-soluble solvent at a temperature of room temperature to 80 ° C., preferably 60 to 70 ° C. to prepare a paramylon solution.
As the paramylon-soluble solvent, an alkaline aqueous solution such as a sodium hydroxide aqueous solution, DMSO (dimethyl sulfoxide), a formic acid aqueous solution, formaldehyde, or the like may be used.
Further, an ionic liquid composed of an imidazolium cation and a halogen or a pseudohalogen anion and a nitrogen-based organic solvent, or a solvent composed of this ionic liquid and DMSO (dimethyl sulfoxide) may be used.
Here, the ionic liquid comprising an imidazolium cation and a halogen or pseudohalogen anion is particularly preferably a compound represented by the chemical structural formula of Formula 1.

In the formula, R ₁ is an alkyl group having 1 to 4 carbon atoms, and R ₂ is an alkyl group having 1 to 4 carbon atoms or an allyl group. X is halogen or pseudohalogen.

Examples of these ionic liquids include 1-butyl-3-methylimidazolium chloride (BMIMCL), 1-butyl-3-methylimidazolium bromide, 1-allyl-3-methylimidazolium chloride, 1-butyl bromide. Examples include allyl-3-methylimidazolium, 1-propyl-3-methylimidazolium bromide, 1-ethyl-3-methylimidazolium acetate, and 1-ethyl-3-methylimidazolium formate.
Examples of the nitrogen-based organic solvent include N, N-dimethylacetamide, N, N-dimethylformamide, 1-methyl-2-pyrrolidone and the like.

The amount of paramylon powder added to an ionic liquid consisting of an imidazolium cation and a halogen or pseudohalogen anion and a nitrogen-based organic solvent, or a solvent consisting of this ionic liquid and DMSO (dimethyl sulfoxide) is such that the paramylon concentration in the paramylon solution is 2 It is preferably in the range of ˜20% by weight.
Moreover, since the decomposition | disassembly of a solvent will begin to occur when the temperature when paramylon is dissolved in a paramylon-soluble solvent exceeds 80 ° C, the temperature is preferably 80 ° C or lower. Moreover, since it takes time for dissolution at a low temperature, 60 to 70 ° C. is preferable.

Next, the paramylon solution is cooled to 40 to 50 ° C. and applied to the glass substrate by a known bar coating method so that the wet film thickness is about 700 μm. Here, the bar coating method is a method in which guides slightly thicker than the substrate are placed on both sides of the substrate, and the gel-like substance hung on the substrate is extended with a bar.
Next, the glass substrate coated with the paramylon solution by the bar coating method is immersed in a coagulant. As the paramylon solution coagulates, the gel film peels off from the substrate. Therefore, the gel film is pulled up and immersed in a new coagulant three times to perform solvent replacement.

The coagulant of the paramylon solution of the present invention is not particularly limited as long as it is a solvent that can extract an ionic liquid and a nitrogen-based organic solvent, but water, acetone, Preferred are ketones such as methyl ethyl ketone, alcohols such as methanol, ethanol and propanol, esters such as methyl acetate, dimethylacetamide, N, N-dimethylformamide and the like. Among these, water, acetone, methanol and ethanol are the present invention. In order to achieve the above-mentioned problem, the solvent is particularly preferably used.
Thereafter, the gel film is attached to a glass substrate and dried at room temperature for 2-3 hours. After drying, the gel film is peeled off from the substrate to obtain a film having a thickness of about 20 μm.

Further, in the above embodiment, the wound healing agent produced in the present invention was formed as a film-like body, but instead of being formed into a film shape by the bar coat method, it is put into a container of a spinning machine having an extrusion function, You may form in a fibrous form by discharging from a nozzle in the coagulant | flocculant of a paramylon solution. Moreover, you may shape | mold into other shapes, such as a pellet form, using an extruder.
Further, together with paramylon derived from Euglena, a conventionally known dosage form may be used as a pharmaceutically acceptable pharmaceutical carrier.
Specifically, it can be a dosage form such as pasta, ointment, cream, liquid, gel, patch, patch, patch, emulsion type, and the like.

The pasta agent can be used in the form of an oily pasta agent, and as the base component, for example, fats, waxes, hydrocarbons and the like are used.
In the case of an ointment, as a base component, for example, fats, polyhydric alcohols, hydrocarbons and the like can be used.
In the case of a cream, for example, a surfactant, a higher alcohol, a higher fatty acid, a hydrocarbon, a polyhydric alcohol, water (purified water) or the like can be used as a base component.

In the case of liquids and gels, for example, water (purified water), lower alcohols, ketones, fats, polyhydric alcohols, surfactants, hydrocarbons, synthetic and natural polymers are used as base components. be able to. However, since paramylon is insoluble in water, when water is used as a base component, a water-soluble paramylon derivative or the like may be used instead of paramylon.
In addition, the wound healing agent produced in the present invention may be applied to the affected area as it is, but for example, a poultice obtained by applying the external preparation to a stretchable cloth, nonwoven fabric, plastic sheet or the like. Or it may be applied to the affected area as a patch such as plaster. Alternatively, an external preparation for wound treatment in a liquid dosage form may be stored in a spray container and sprayed onto the affected area.

The wound healing process has mutually overlapping periods of an inflammatory phase, a proliferative phase, and a mature phase, and in the inflammatory phase, neutrophils and monocytes / macrophages that wet the damaged part play an important role. The expression level of IL-10 protein in the damaged skin area of the mouse reaches a peak in the early inflammatory period (3 hours after the injury) in which the neutrophils that migrate to the damaged area gradually increase, and then decreases once. There is a report that peak is reached again at the extreme stage of inflammation (72 hours after injury) when wrinkles are most prominent, and mRNA expression correlates well with this (Yasunori Sato, “Interleukin-10 in skin wound healing process. Expression and its forensic application ", Kanazawa University Juzen Medical Community Journal, Vol. 108, No. 4, p.486-502). Here, IL-10 is an anti-inflammatory cytokine.
Specific periods in each period include, for example, the inflammatory phase is 0 to 3 days after injury, the proliferative phase is 3 to 2 weeks after injury, the mature phase is 2 weeks to several months or years after injury, etc. It is said.

And by our experiments, it has been found that Euglena-derived paramylon acts particularly on neoplastic and inflammatory macrophages and promotes inflammatory cytokine secretion. Examples of inflammatory cytokines include TNF-α, IFN-α, IFN (interferon) -γ, and IL-6.
Therefore, the therapeutic agent for wounds produced in the present invention is preferably applied to the wound at least from immediately after the wound is injured to the inflammatory stage in the healing process.
By being applied in this way, secretion of cytokines is efficiently promoted in the early inflammatory phase, the extreme inflammatory phase, etc., and the wound healing promoting effect is improved.
Moreover, the wound therapeutic agent produced by this invention may be used as a wound therapeutic agent for diabetic patients applied to the wound of diabetic patients whose cell migration becomes dull and secretion of cytokines is suppressed.

EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited only to these.
<Example 1: Production of paramylon film>
10 parts of paramylon powder is added to 100 parts of a mixed solvent obtained by mixing 1-butyl-3-methylimidazolium chloride (BMIMCL) and dimethylacetamide in a ratio of 5: 2, and the paramylon powder is dissolved at 60 to 70 ° C. Thus, a paramylon solution was obtained. This solution was cooled to 40 to 50 ° C. and applied to a glass substrate by a bar coating method so as to have a wet film thickness of about 700 μm. Thereafter, the coated material was immersed in a methanol bath together with the glass substrate. As the paramylon solution was solidified, the gel film was peeled off from the substrate, so that the gel film was pulled up and immersed in fresh methanol three times to perform solvent replacement. Thereafter, the gel film was attached to a glass substrate and dried at room temperature for 2 to 3 hours. After drying, the film was peeled from the substrate to obtain a paramylon film having a thickness of about 20 μm.

<Test Example 1: Examination of Wound Healing Promoting Action by Paramylon Film of Example 1>
The anti-inflammatory action and the wound healing promoting action of the paramylon film of Example 1 were examined by evaluating the repair of the defect wound produced on the back of the mouse. This Test Example 1 was conducted according to the experimental protocol shown in FIG. 1, and water and feed were freely consumed during the acclimatization period and the main test period. Mouse M was bred at a room temperature of 22 ± 1 ° C. and a light / dark cycle of 12 hours (light cycle: 8: 00-20: 00).
In addition, Test Example 1 was conducted in accordance with standards (2006: Ministry of the Environment notification) concerning the breeding and storage of experimental animals.

1. Preparation of Wounds and Fixation of Various Films First, 8-week-old male Jcl: ICR mice (Claire Japan) were acclimatized for 4 days with feed for breeding CE-2 (Claire Japan).
Next, mouse M was anatomized with chloral hydrate (400 mg / kg: Wako Pure Chemical Industries, Ltd., Tokyo) using a clipper and a hair removal gel mousse (Rekit Benkieser Japan Co., Ltd., Tokyo). The back was extensively depilated. The mouse M was laid sideways and piled while pulling the skin on the back, and the epithelium was cut off with a biopsy punch (diameter 8 mm) to produce a wound 1 having a diameter of 8 mm as shown in FIG.
For the mice of Example 1, the wound was filled with the paramylon film F of Example 1 having a diameter of 8 mm by biopsy punch into the wound, and dressing film 2 (Johnson & Johnson Co., Ltd., Tokyo, below) And the fixing was performed as shown in FIG.

Moreover, about the mouse | mouth of the comparative example 1 group, the cellulose film F of the comparative example 1 made into the circle of diameter 8mm with a biopsy punch about the wound 1 (cellophane tube for dialysis 23.8 (phi) x5m, molecular weight cut-off 10,000- 14,000: Kennis Co., Ltd., Osaka) was embedded in the wound and fixed using dressing film 2 as shown in FIG.
The mouse | mouth of the comparative example 2 group is a control group, nothing was apply | coated to the wound 1, and it fixed as shown in FIG. 2 using the adhesive bandage and the dressing film 2. FIG.

2. Observation of wound healing process For each group in which wounds were prepared in 0, 1, 3, 5, and 7 days after wounding, the body weight of the mice, the wound healing area, and on days 0, 1, 3, and 5 The size of the raised part of the dermis around the mouse wound was evaluated.
As the degree of anti-inflammatory healing in each group, the daily wound healing area was calculated using the wound area ratio.
Wound area ratio is
Wound area ratio (%) = A / B × 100
However, A: The major axis × minor axis of the defect wound on each measurement date B: The major axis × minor axis of the defect wound on the defect creation date was calculated.

In addition, the daily dermal area around the wound was calculated as the size of the raised portion of the dermis around the wound.
The dermis area around the wound is
Wound peripheral dermis area (mm ² ) = C−A
However, C: The major axis × minor axis of the wound peripheral dermis on each measurement day A: Calculated by the major axis × minor axis of the defect wound on each measurement day.
Since it is empirically known that wound healing is promoted when the dermis around the wound is large, in this test example, the dermal swell around the wound of each group of mice It was decided to evaluate the size.

  For data aggregation and analysis, 4steps Excel (registered trademark) Statistics Statcel 3 (OMS Publishing, Saitama, Ltd.), which is a statistical analysis program, was used. After confirming the normalization of each group, the analysis was performed.

  Analysis of variance (ANOVA: two-way layout) and multiple comparison test (Tukey-Kramer) were used to test for differences within each group and between groups. The significance level was less than 5%, and all analysis values were expressed as mean ± standard deviation.

FIG. 3 shows the measurement results of the weights of the mice in each group on days 0, 1, 3, 5, 7, and 10. As shown in FIG. 3, there was no significant change in body weight between groups throughout the experimental period.
FIG. 4 shows the measurement results of the wound area ratios of the mice in each group at 0, 1, 3, 5, 7, and 10 days. As shown in FIG. 4, the wound area ratio was decreased in each group on days 3, 5, 7, and 10 as compared with day 0 of wound creation. In particular, in the paramylon film group of Example 1, it decreased remarkably with time.
FIG. 5 shows the measurement results of the dermis area around the wound of each group of mice on days 0, 1, 3, 5, 7, and 10. As shown in FIG. 5, the dermis area around the wound was not changed in each group until the third day. However, on the fifth day, a marked increase was observed in the paramylon film group of Example 1, which was twice the area of the Comparative Example 1 group and the Comparative Example 2 group. On the 10th day after becoming smaller than Example 1, it was the same area as Comparative Examples 1 and 2.
In addition, Table 1 shows the wound area ratio and the dermal area around the wound of the mice of each group on the 5th and 7th days.

Therefore, in Example 1 group, as shown in FIG. 5, healing in the inflammatory stage progresses in the wound healing process earlier than Comparative Examples 1 and 2, and as shown in FIG. It was found that the area of the wound itself was smaller than those of Comparative Examples 1 and 2.
In Example 1, the dermis was formed earlier and the wound was healed. The Example 1 group was found to be close to natural healing and to promote healing without imposing a burden on the body.

FIGS. 6 to 9 are photographs of wounds around each group on day 0, day 1, day 3, and day 5 after wound preparation, respectively. By visual observation, it was observed that the dermis around the wound was greatly raised only in the paramylon film group of Example 1 one day after wound preparation.
From the above, as shown in FIGS. 6 and 7, in comparison with Comparative Examples 1 and 2, the paramylon film group of Example 1 was compared with Comparative Examples 1 and 2 after 0 and 1 day after wound preparation. As shown in FIGS. 7 to 9, at the same time that the surrounding dermis is greatly swelled, as shown in FIGS. 7 to 9, Comparative Example 1 was observed from 1 day to 5 days after wound preparation, especially 5 days later. , 2, the dermal area around the wound was significantly larger.
Further, in the paramylon film group of Example 1, the wound healing area was also compared with Comparative Examples 1 and 2 from 1 day to 7 days after wound preparation, particularly from 5 days to 7 days later. Was significantly smaller.

  It is empirically known that healing of the wound is promoted when the dermis around the wound is large. Also in the paramylon film group of Example 1, as shown in FIGS. 6 and 7, it was observed that the dermis around the wound was greatly raised 0, 1 day after wound preparation, and thereafter, in FIGS. 7 to 9. As shown, wound healing was greatly accelerated.

3. Measurement of blood cytokine level by ELISA method 1. The amount of IL-6, IFN-γ, and VEGF in the blood of each group of mice that produced wounds in (1) was measured by ELISA (sandwich method).
Above 1. For each group in which wounds were prepared in step 1, the blood of mice on day 0, day 3, and day 5 after wound preparation was used as an ELISA sample.
For the measurement, an ELISA Kit manufactured by PeproTech was used.
The measurement results of IL-6 amount, IFN-γ amount, and VEGF amount are shown in FIGS. 10, FIG. 12 and FIG. 14 show the blood IL-6 amount, the blood IFN-γ amount, and the blood VEGF amount after 0 days, 3 days and 5 days after wound creation in the control group of Comparative Example 2. 11, FIG. 13 and FIG. 15 show blood IL-6 after 3 days and 5 days after wound preparation in the paramylon film group of Example 1, the cellulose film group of Comparative Example 1, and the control group of Comparative Example 2. Amount, blood IFN-γ amount, blood VEGF amount are shown.

12 and 13, in Comparative Examples 1 and 2, the amount of IFN-γ decreased after 3 days, but in Example 1, the amount of IFN-γ after 3 days was large, and the paramylon film group of Example 1 There was a tendency that blood IFN-γ was expressed in the late inflammatory period, which is the initial stage of healing, and then decreased.
IFN-γ is a cytokine that contributes to the activation of neoplastic and inflammatory macrophages and is over-released during the inflammatory phase. In the inflammatory phase, vasodilation and increased vascular permeability occur due to excessive release of inflammatory cytokines including IFN-γ, histamine, prostacyclin and the like.
In Example 1, as shown in FIG. 13, IFN-γ, which is one of inflammatory cytokines, was significantly expressed even in comparison with Comparative Examples 1 and 2 in the early stage of healing. This tendency is shown in 2. , The dermis around the wound greatly swelled during the inflammatory phase, which is the initial stage of healing, and was consistent with the result that the healing of the wound was greatly promoted thereafter.

F film (paramylon film, cellulose film)
M Mouse 1 Wound 2 Dressing film

Claims (1)

A paramylon solution obtained by dissolving paramylon derived from Euglena in a paramylon-soluble solvent at a temperature of 60 to 70 ° C. is cooled to 40 to 50 ° C. , and then extracted as a poor solvent for the paramylon and the paramylon-soluble solvent. method for producing passed through a solvent capable to wound treatment agent wounds you characterized that you produce paramylon moldings.